Stimulus-inducible protein kinase complex and methods of use therefor

ABSTRACT

Compositions and methods are provided for treating NF-κB-related conditions. In particular, the invention provides a stimulus-inducible IKK signalsome, and components and variants thereof. An IKK signalsome or component thereof may be used, for example, to identify antibodies and other modulating agents that inhibit or activate signal transduction via the NF-κB cascade. IKK signalsome, components thereof and/or modulating agents may also be used for the treatment of diseases associated with NF-κB activation.

CROSS-REFERENCE TO PRIOR APPLICATION

This application is a divisional application of Ser. No. 09/844,908,filed Apr. 27, 2001, now U.S. Pat. No. 6,576,437 which is a divisionalapplication of Ser. No. 08/910,820, filed Aug. 13, 1997 now U.S. Pat.No. 6,258,579, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/697,393, filed Aug. 26, 1996 now U.S. Pat No.5,972,674.

TECHNICAL FIELD

The present invention relates generally to compositions and methodsuseful for the study of cascades leading to the activation of nuclearfactor κB (NF-κB) and for treating diseases associated with suchpathways. The invention is more particularly related to astimulus-inducible IκB kinase (IKK) signalsome, component IκB kinasesand variants of such kinases. The present invention is also related tothe use of a stimulus-inducible IKK signalsome or IκB kinase to identifyantibodies and other agents that inhibit or activate signal transductionvia the NF-κB pathway.

BACKGROUND OF THE INVENTION

Transcription factors of the NFκB/Rel family are critical regulators ofgenes involved in inflammation, cell proliferation and apoptosis (forreviews, see Verma et al., Genes Dev. 9:2723-35, 1995; Siebenlist,Biochim. Biophys. Acta 1332:7-13, 1997, Baeuerle and Henkel, Ann. Rev.Immunol. 12:141-79, 1994; Barnes and Karin, New Engl. J. Med. 336,1066-71, 1997; Baeuerle and Baltimore, Cell 87:13-20, 1996; Grilli etal., NF-κB and Rel: Participants in a multiform transcriptionalregulatory system (Academic Press, Inc., 1993), vol. 143; Baichwal andBaeuerle, Curr. Biol. 7:94-96, 1997). The prototype member of thefamily, NFκB, is composed of a dimer of p50 NFκB and p65 RelA (Baeuerleand Baltimore, Cell 53:211-17, 1988; Baeuerle and Baltimore, Genes Dev.3:1689-98, 1989). NF-κB plays a pivotal role in the highly specificpattern of gene expression observed for immune, inflammatory and acutephase response genes, including interleukin 1, interleukin 8, tumornecrosis factor and certain cell adhesion molecules.

Like other members of the Rel family of transcriptional activators,NF-κB is sequestered in an inactive form in the cytoplasm of most celltypes. A variety of extracellular stimuli including mitogens, cytokines,antigens, stress inducing agents, UV light and viral proteins initiate asignal transduction pathway that ultimately leads to NF-κB release andactivation. Thus, inhibitors and activators of the signal transductionpathway may be used to alter the level of active NF-κB, and havepotential utility in the treatment of diseases associated with NF-κBactivation.

Activation of NFκB in response to each of these stimuli is controlled byan inhibitory subunit, IκB, which retains NFκB in the cytoplasm. IκBproteins, of which there are six known members, each contain 5-7ankyrin-like repeats required for association with the NFκB/Rel dimerand for inhibitory activity (see Beg et al., Genes Dev. 7, 2064-70,1993; Gilmore and Morin, Trends Genet. 9, 427-33, 1993; Diaz-Meco etal., Mol. Cell. Biol. 13:4770-75, 1993; Haskill et al., Cell 65:1281-89,1991). IκB proteins include IκBα and IκBβ.

NFκB activation involves the sequential phosphorylation, ubiquitination,and degradation of IκB. Phosphorylation of IκB is highly specific fortarget residues. For example, phosphorylation of the IκB protein IκBαtakes place at serine residues S32 and S36, and phosphorylation of IκBβoccurs at serine residues S19 and S23. The choreographed series ofmodification and degradation steps results in nuclear import oftranscriptionally active NFκB due to the exposure of a nuclearlocalization signal on NFκB that was previously masked by IκB (Beg etal., Genes Dev. 6:1899-1913, 1992). Thus, NFκB activation is mediated bya signal transduction cascade that includes one or more specific IκBkinases, a linked series of E1, E2 and E3 ubiquitin enzymes, the 26Sproteasome, and the nuclear import machinery. The phosphorylation of IκBis a critical step in NF-κB activation, and the identification of an IκBkinase, as well as proteins that modulate its kinase activity, wouldfurther the understanding of the activation process, as well as thedevelopment of therapeutic methods.

Several protein kinases have been found to phosphorylate IκB in vitro,including protein kinase A (Ghosh and Baltimore, Nature 344:678-82,1990), protein kinase C (Ghosh and Baltimore, Nature 344:678-82, 1990)and double stranded RNA-dependent protein kinase (Kunar et al., Proc.Natl. Acad. Sci. USA 91:6288-92, 1994). Constitutive phosphorylation ofIκBα by casein kinase II has also been observed (see Barroga et al.,Proc. Natl. Acad. Sci. USA 92:7637-41, 1995). None of these kinases,however appear to be responsible for in vivo activation of NF-κB. Forexample, phosphorylation of IκBα in vitro by protein kinase A andprotein kinase C prevent its association with NF-κB, and phosphorylationby double-stranded RNA-dependent protein kinase results in dissociationof NF-κB. Neither of these conform to the effect of phosphorylation invivo, where IκBα phosphorylation at S32 and S36 does not result indissociation from NF-κB.

Other previously unknown proteins with IκB kinase activity have beenreported, but these proteins also do not appear to be significantactivators in vivo. A putative IκBα kinase was identified by Kuno etal., J. Biol. Chem. 270:27914-27919, 1995, but that kinase appears tophosphorylate residues in the C-terminal region of IκBα, rather than theS32 and S36 residues known to be important for in vivo regulation.Diaz-Meco et al., EMBO J. 13:2842-2848, 1994 also identified a 50 kD IκBkinase, with uncharacterized phosphorylation sites. Schouten et al.,EMBO J. 16:3133-44, 1997 identified p90^(rski) as a putative IκBαkinase; however, p90^(rski) is only activated by TPA and phosphorylatesIκBα only on Ser32, which is insufficient to render IκBα a target forubiquitination. Finally, Chen et al, Cell 84:853-862, 1996 identified akinase that phosphorylates IκBα, but that kinase was identified using anon-physiological inducer of IκBα kinase activity and requires theaddition of exogenous factors for in vitro phosphorylation.

Accordingly, there is a need in the art for an IκB kinase that possessesthe substrate specificity and other properties of the in vivo kinase.There is also a need for improved methods for modulating the activity ofproteins involved in activation of NF-κB, and for treating diseasesassociated with NF-κB activation. The present invention fulfills theseneeds and further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsemploying a large, multi-subunit IKK signalsome, or a component orvariant thereof. In one aspect, the present invention provides an IKKsignalsome capable of specifically phosphorylating IκBα at residues S32and S36, and IκBβ at residues 19 and 23, without the addition ofexogenous cofactors.

In a further related aspect, a polypeptide comprising a component of anIKK signalsome, or a variant of such a component, is provided, whereinthe component has a sequence recited in SEQ ID NO:9. An isolated DNAmolecule and recombinant expression vector encoding such a polypeptide,as well as a transfected host cell, are also provided.

In another aspect, methods for preparing an IKK signalsome are provided,comprising combining components of an IKK signalsome in a suitablebuffer.

In yet another aspect, methods are provided for phosphorylating asubstrate of an IKK signalsome, comprising contacting a substrate withan IKK signalsome or a component thereof, and thereby phosphorylatingthe substrate.

In a further aspect, the present invention provides a method forscreening for an agent that modulates IKK signalsome activity,comprising: (a) contacting a candidate agent with an IKK signalsome,wherein the step of contacting is carried out under conditions and for atime sufficient to allow the candidate agent and the IKK signalsome tointeract; and (b) subsequently measuring the ability of the candidateagent to modulate IKK signalsome activity.

Within a related aspect, the present invention provides methods forscreening for an agent that modulates IKK signalsome activity,comprising: (a) contacting a candidate agent with a polypeptidecomprising a component of an IKK signalsome as described above, whereinthe step of contacting is carried out under conditions and for a timesufficient to allow the candidate agent and the polypeptide to interact;and (b) subsequently measuring the ability of the candidate agent tomodulate the ability of the polypeptide to phosphorylate an IκB protein.

In another aspect, an antibody is provided that binds to a component(e.g., IKK-1 and/or IKK-2) of an IKK signalsome, where the component iscapable of phosphorylating IκBα.

In further aspects, the present invention provides methods formodulating NF-κB activity in a patient, comprising administering to apatient an agent that modulates IκB kinase activity in combination witha pharmaceutically acceptable carrier. Methods are also provided fortreating a patient afflicted with a disorder associated with theactivation of IKK signalsome, comprising administering to a patient atherapeutically effective amount of an agent that modulates IκB kinaseactivity in combination with a pharmaceutically acceptable carrier.

In yet another aspect, a method for detecting IKK signalsome activity ina sample is provided, comprising: (a) contacting a sample with anantibody that binds to an IKK signalsome under conditions and for a timesufficient to allow the antibody to immunoprecipitate an IKK signalsome;(b) separating immunoprecipitated material from the sample; and (c)determining the ability of the immunoprecipitated material tospecifically phosphorylate an IκB protein with in vivo specificity.Within one such embodiment, the ability of the immunoprecipitatedmaterial to phosphorylate IκBα at residues S32 and/or S36 is determined.

In a related aspect, a kit for detecting IKK signalsome activity in asample is provided, comprising an antibody that binds to an IKKsignalsome in combination with a suitable buffer.

In a further aspect, the present invention provides a method foridentifying an upstream kinase in the NF-κB signal transduction cascade,comprising evaluating the ability of a candidate upstream kinase tophosphorylate an IKK signalsome, a component thereof or a variant ofsuch a component.

A method for identifying a component of an IKK signalsome is alsoprovided, comprising: (a) isolating an IKK signalsome; (b) separatingthe signalsome into components, and (c) obtaining a partial sequence ofa component.

In yet another aspect, a method is provided for preparing an IKKsignalsome from a biological sample, comprising: (a) separating abiological sample into two or more fractions; and (b) monitoring IκBkinase activity in the fractions.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are autoradiograms depicting the results of immunoblotanalyses. FIG. 1A shows the recruitment of IκBα into a high molecularweight complex upon stimulation. Cytoplasmic extracts of eitherunstimulated or PMA (50 ng/ml)- and PHA (1 μg/ml)-stimulated (10 min)Jurkat cells were fractionated on a gel filtration column. IκBα wasvisualized by immunoblot analysis. The upper panel shows the elutionprofile of unstimulated cells, and the lower panel shows the elutionprofile of PMA/PHA-stimulated cells. Lanes 1-14 are successive elutionfractions. Molecular weight standards are indicated by arrows on thetop.

FIG. 1B shows that the stimulus-dependent IκBα kinase activitychromatographs as a high molecular weight complex, M_(T) 500-700 kDa.Whole cell extract of TNFα-stimulated (20 ng/ml, 7 min) HeLa S3 cellswas fractionated on a Superdex 200 gel filtration column and monitoredfor IκBα kinase activity. Lanes 4-10 are successive elution fractions.Phosphorylation of the GST IκBα 1-54 (wildtype) substrate is indicatedby an arrow to the right. Molecular weight standards are indicated byarrows on the top.

FIG. 1C illustrates the identification of proteins that co-chromatographwith the IKK signalsome. IKK signalsome was partially purified fromextracts of TNFα-stimulated HeLa S3 cells by sequential fractionation ona Q Sepharose, Superdex 200, Mono Q, and Phenyl Superose columns. PhenylSuperose fractions containing the peak of IKK signalsome activity weresubjected to western blot analysis using several different antibodies asindicated to the left of each respective panel. The level of IKKsignalsome activity is indicated in the upper shaded area by increasingnumber of (+)'s.

FIG. 2 is a flow chart depicting a representative purification procedurefor the preparation of an IKK signalsome.

FIGS. 3A and 3B are autoradiograms that show the results of a Westernblot analysis of the levels of IκBα in HeLa S3 cytoplasmic extractsfollowing gel filtration. The extracts were prepared from cells thatwere (FIG. 3B) and were not (FIG. 3A) exposed to TNFα. X axis:successive gel filtration fractions.

FIGS. 4A and 4B are autoradiograms depicting the results of an in vitrokinase assay in which the ability of the above cell extracts tophosphorylate the N-terminal portion of IκBα was evaluated. FIG. 4Ashows the results employing an extract from cells that were not treatedwith TNFα, and FIG. 4B shows the results when the cells were treatedwith TNFα. X axis: successive gel filtration fractions with and withoutTNFα.

FIGS. 5A and 5B are autoradiograms depicting the results of an in vitrokinase assay using a cytoplasmic extract of TNFα-treated HeLa S3 cells,where the extract is subjected to Q Sepharose fractionation. Thesubstrate was the truncated IκBα (residues 1 to 54), with FIG. 5Ashowing the results obtained with the wild type IκBα sequence and FIG.5B presenting the results obtained using a polypeptide containingthreonine substitutions at positions 32 and 36. X axis: successivechromatography fractions eluted with a sodium chloride buffer gradient.

FIGS. 6A and 6B are autoradiograms depicting the results of an in vitrokinase assay using a cytoplasmic extract of TNFα-treated HeLa S3 cells,where the extract was subjected in series to chromatographicfractionation by Q Sepharose, Superdex 200, Mono Q Sepharose and PhenylSuperose. The substrate was the truncated IκBα (residues 1 to 54), withFIG. 6A showing the results obtained with the wild type IκBα sequenceand FIG. 6B presenting the results obtained using a polypeptidecontaining threonine substitutions at positions 32 and 36. X axis:successive chromatography fractions eluted with an ammonium sulfatebuffer gradient.

FIG. 7 is an autoradiogram showing the results of immunokinase assays(using anti-MKP-1 antibody) performed using cytoplasmic extracts ofTNFα-treated HeLa S3 cells following gel filtration. The assay wasperformed using the substrates GST-IκBα1-54 wildtype (lane 1) andGST-IκBα1-54 S32/36 to T (lane 2). The positions of IκBα and GST-IκBα1-54 are indicated on the left.

FIGS. 8A-8C are autoradiograms depicting the results of immunoblotanalyses. In FIG. 8A, the upper panel presents a time course for theinduction of signalsome activity. Anti MKP-1 immune precipitates fromextracts of HeLa S3 cells stimulated with TNFα (20 ng/ml) for theindicated times, were assayed for IKK signalsome activity by standardimmune complex kinase assays. 4 μg of either GST IκBα 1-54 WT (wildtype)or the GST IκBα 1-54 S32/36 to T mutant (S>T) were used as thesubstrates. In the lower panel, HeLa cell extracts prepared as describedin the upper panel were examined by western blot analysis for IκBαdegradation. IκBα supershifting phosphorylation can be seen after 3 and5 minutes of stimulation followed by the disappearance of IκBα.

FIG. 8B illustrates the stimulus-dependent activation of IKK signalsome,which is blocked by TPCK. Anti-MKP-1 immunoprecipitates from cellextracts of HeLa S3 cells either stimulated for 7 min with TNFα (20ng/ml, lane 2 and 6), IL-1 (10 ng/ml, lane 3), PMA (50 ng/ml, lane 4) orpretreated for 30 min with TPCK (15 μM, lane 7) prior to TNFα-inductionwere examined for IKK signalsome activity. GST IκBα 1-54 WT (4 μg) wasused as a substrate.

FIG. 8C illustrates the ability of IKK signalsome to specificallyphosphorylate serines 32 and 36 of the IκBα holoprotein in the contextof a RelA:IκBα complex. Anti-MKP-1 immunoprecipitates from cell extractsof HeLa S3 cells stimulated with TNFα (20 ng/ml, 7 min) were examinedfor their ability to phosphorylate baculoviral expressed RelA:IκBαcomplex containing either the IκBα WT (lane 3) or IκBα S32/36 to Amutant (lane 4) holoprotein. The specific substrates used are indicatedon the top. Positions of the phosphorylated substrates are indicated byarrows to the left of the panel.

FIG. 9A is an autoradiogram depicting the results of an immunokinaseassay in which peptides are phosphorylated by the IKK signalsome. In thetop panel, IκBα (21-41) peptides that were unphosphorylated orchemically phosphorylated on either Ser-32 or Ser-36 were incubated withthe IKK signalsome in the presence of γ-[³²P]-ATP. The doublyphosphorylated peptide P32,36 was not phosphorylated by the IKKsignalsome, and the unrelated c-Fos(222-241) phosphopeptide with freeserine and threonine residues did not function as a signalsomesubstrate.

FIG. 9B is a graph illustrating the inhibition of phosphorylation ofGST-IκBα (1-54) by IκBα (21-41) peptides. IκBα (21-41) peptide P32,36inhibits GST-IκBα (1-54) as a product inhibitor with a K_(i) value of 14μM. The unrelated phosphopeptide c-Fos(222-241) does not function as aninhibitor. This assay only detects precipitated ³²P-labeled proteins,not ³²P-labeled peptides. Addition of the singly- or non-phosphorylatedIκBα (21-41) peptides results in less phosphorylation of GST-IκBα (1-54)and apparent inhibition.

FIG. 10 is an autoradiogram showing the results of a western blotanalysis of the level of ubiquitin within a stimulus-inducible IkBkinase complex. Lane 1 shows the detection of 100 ng ubiquitin, Lane 2shows 10 ng ubiquitin and Lane 3 shows 3.4 μg of IKK signalsome purifiedthrough the phenyl superose step (sufficient quantities for 10 kinasereactions). The position of ubiquitin is shown by the arrow on the left.

FIG. 11A illustrates a procedure for purification of the IKK signalsome.A whole cell extract was prepared from TNFα-stimulated (20 ng/ml, 7minute induction) HeLa S3 cells (1.2 g total protein). The IKKsignalsome was then immunoprecipitated from the extract using anti-MKP-1antibodies, washed with buffer containing 3.5 M urea and elutedovernight at 4° C. in the presence of excess MKP-1 specific peptide.Eluted IKK signalsome was then fractionated on a Mono Q column, IκBkinase active fractions were pooled, concentrated and subjected topreparative SDS-PAGE. Individual protein bands were excised andsubmitted for peptide sequencing.

FIG. 11B is a photograph showing Mono Q fractions containing active IKKsignalsome activity following SDS-PAGE and a standard silver stainprotocol. Peak activity of IKK signalsome activity is represented inlanes 3, 4, and 5. Protein bands corresponding to IKK-1 and IKK-2 areindicated to the left of the figure. Molecular weight standards (kDa)are indicated to the left of the figure.

FIGS. 12A and 12B are mass spectra obtained during sequencing of IKK-2by nanoelectrospray mass spectrometry. FIG. 12A shows part of the massspectrum of the unseparated mixture of tryptic peptides resulting fromin-gel digestion of the IKK-2 band in FIG. 11B. FIG. 12B shows a tandemmass spectrum of the peak at m/z 645.2.

FIG. 13A illustrates the amino acid sequence of IKK-1 (SEQ ID NO: 10)and IKK-2 (SEQ ID NO:9). Symbols: arrows, boundaries of the kinasedomain; underlined, peptide sequences identified by nanoelectrospraymass spectrometry; asterisks, indicates leucines comprising the leucinezipper motif; bold face, indicate amino acid identities conservedbetween IKK-1 and IKK-2; highlighted box, Helix-loop-helix domain;dashes, a gap inserted to optimize alignment.

FIG. 13B is an autoradiogram depicting the results of Northern blotanalysis of IKK-2 mRNA in adult human tissue. The source of the tissueis indicated at the top. Probes spanning the coding region of humanIKK-2 and β-actin cDNA were used and are indicated to the left.Molecular weight standards are indicated to the right.

FIG. 14A is an autoradiogram depicting the results of kinase assaysusing IKK-1 and IKK-2. IKK-1 and IKK-2 were immunoprecipitated fromrabbit reticulocyte lysates phosphorylate IκBα and IκBβ. EitherHA-tagged IKK-1 (lane 1) or Flag-tagged IKK-2 (lane 2) were translatedin rabbit reticulocyte lysates, immunoprecipitated, and examined fortheir ability to phosphorylate GST IκBα 1-54 WT and GST IκBβ 1-44 asindicated by an arrow to the left. IKK-1 (lane 1) undergoes significantautophosphorylation in contrast to IKK-2 (lane 2) which is identifiedonly with longer exposure times.

FIGS. 14B and 14C are micrographs illustrating the results of assays toevaluate the ability of kinase-inactive mutants of IKK-1 and IKK-2 toinhibit RelA translocation in TNFα-stimulated HeLa cells. HeLa cellswere transiently transfected with either HA-tagged IKK-1 K44 to M mutant(14B) or Flag-tagged IKK-2 K44 to M mutant (14C) expression vectors. 36hours post-transfection cells were either not stimulated (Unstim) orTNFα-stimulated (20 ng/ml) for 30 min (TNFα), as indicated to the rightof the figure. Cells were then subjected to immunofluorescence stainingusing anti-HA of anti-Flag antibodies to visualize expression of IKK-1K44 to M or IKK-2 K44 to M, respectively. Stimulus-dependenttranslocation of Rel A was monitored using anti-Rel A antibodies.Antibodies used are indicated to the top of the figure. IKK mutanttransfected is indicated to the left of the figure.

FIGS. 15A and 15B are autoradiograms of immunoprecipitated IKK-1 andIKK-2 following in vitro translation. In FIG. 15A, HA-tagged IKK-1 andFlag-tagged IKK-2 were in vitro translated in wheat germ lysates eitherseparately or in combination, as indicated. The programmed translationmix was then subjected to immunoprecipitation using the indicatedantibody. The samples were run on SDS-PAGE and subjected toautoradiography. In FIG. 15B, HA-tagged IKK-1 and Flag-tagged IKK-2 werein vitro translated in rabbit reticulocyte lysates either separately orin combination, as indicated. The programmed translation mix was thensubjected to immunoprecipitation using the indicated antibody. Thesamples were run on SDS-PAGE and subjected to autoradiography. Theresults show that IKK-1 and IKK-2 coprecipitate when translated inrabbit reticulocyte lysates.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for modulating (i.e., stimulating orinhibiting) signal transduction leading to NF-κB activation. Inparticular, the present invention is directed to compositions comprisingan IκB kinase (IKK) signalsome (also referred to herein as a“stimulus-inducible IκB kinase complex” or “IκB kinase complex”) that iscapable of stimulus-dependent phosphorylation of IκBα and IκBβ on thetwo N-terminal serine residues critical for the subsequentubiquitination and degradation in vivo. Such stimulus-dependentphosphorylation may be achieved without the addition of exogenouscofactors. In particular, an IKK signalsome specifically phosphorylatesIκBα (SEQ ID NO:1) at residues S32 and S36 and phosphorylates IκBβ (SEQID NO:2) at residues S19 and S23. The present invention also encompassescompositions that contain one or more components of such an IKKsignalsome, or variants of such components. Preferred components,referred to herein as “IKK signalsome kinases” “IκB kinases” or IKKs)are kinases that, when incorporated into an IKK signalsome, are capableof phosphorylating IκBα at S32 and S36. Particularly preferredcomponents are IKK-1 (SEQ ID NO:10) and IKK-2 (SEQ ID NO:9).

An IKK signalsome and/or IκB kinase may generally be used forphosphorylating a substrate (i e., an IκB, such as IκBα, or a portion orvariant thereof that can be phosphorylated at those residues that arephosphorylated in vivo) and for identifying modulators of IκB kinaseactivity. Such modulators and methods employing them for modulating IκBαkinase activity, in vivo and/or in vitro, are also encompassed by thepresent invention. In general, compositions that inhibit IκB kinaseactivity may inhibit IκB phosphorylation, or may inhibit the activationof an IκB kinase and/or IKK signalsome.

An IKK signalsome has several distinctive properties. Such a complex isstable (i.e., its components remain associated following purification asdescribed herein) and has a high-molecular weight (about 500-700 kD, asdetermined by gel filtration chromatography). As shown in FIGS. 3(A andB) and 4(A and B), IκB kinase activity of an IKK signalsome is“stimulus-inducible” in that it is stimulated by TNFα (i.e., treatmentof cells with TNFα results in increased IκB kinase activity and IκBdegradation) and/or by one or more other inducers of NF-κB, such asIL-1, LPS, TPA, UV irradiation, antigens, viral proteins andstress-inducing agents. The kinetics of stimulation by TNFα correspondto those found in vivo. IκB kinase activity of an IKK signalsome is alsospecific for S32 and S36 of IκBα. As shown in FIGS. 5(A and B) and 6(Aand B), an IKK signalsome is capable of phosphorylating a polypeptidehaving the N-terminal sequence of IκBα (GST-IκBα1-54; SEQ ID NO:3), butsuch phosphorylation cannot be detected in an IκBα derivative containingthreonine substitutions at positions 32 and 36. In addition, IκB kinaseactivity is strongly inhibited by a doubly phosphorylated IκBα peptide(i.e., phosphorylated at S32 and S36), but not by an unrelated c-fosphosphopeptide that contains a single phosphothreonine. A furthercharacteristic of an IKK signalsome is its ability to phosphorylate asubstrate in vitro in a standard kinase buffer, without the addition ofexogenous cofactors. Free ubiquitin is not detectable in preparations ofIKK signalsome (see FIG. 10), even at very long exposures. Accordinglyan IKK signalsome differs from the ubiquitin-dependent IκBα kinaseactivity described by Chen et al., Cell 84:853-62, 1996.

An IKK signalsome may be immunoprecipitated by antibodies raised againstMKP-1 (MAP kinase phosphatase-1; Santa Cruz Biotechnology, Inc., SantaCruz, Calif. #SC-1102), and its activity detected using an in vitro IκBαkinase assay. However, as discussed further below, MKP-1 does not appearto be a component of IκB kinase complex. The substrate specificity ofthe immunoprecipitated IKK signalsome is maintained (i.e., there isstrong phosphorylation of wildtype GST-IκBα 1-54 (SEQ ID NO:3) andGST-IκBβ 1-44 (SEQ ID NO:4), and substantially no detectablephosphorylation of GST-IκBα 1-54 in which serines 32 and 36 are replacedby threonines (GST-IκBα S32/36 to T; SEQ ID NO:5) or GST-IκBβ 1-44 inwhich serines 19 and 23 are replaced by alanines (GST-IκBβ 1-44 S19/23to A; SEQ ID NO:6)).

An IKK signalsome may be isolated from human or other cells, and fromany of a variety of tissues and/or cell types. For example, usingstandard protocols, cytoplasmic and/or nuclear/membrane extracts may beprepared from HeLa S3 cells following seven minutes induction with 30ng/mL TNFα. The extracts may then be subjected to a series ofchromatographic steps that includes Q Sepharose, gel filtration (HiLoad16/60 Superdex 200), Mono Q, Phenyl Superose, gel filtration (Superdex200 10/30) and Mono Q. This representative purification procedure isillustrated in FIG. 2, and results in highly enriched IKK signalsome(compare, for example, FIGS. 5A and 6A).

An alternative purification procedure employs a two-step affinitymethod, based on recognition of IKK signalsome by the MKP-1 antibody(FIG. 11A). Whole cell lysates from TNFα-stimulated HeLa cells may beimmunoprecipitated with an anti-MKP-1 antibody. The IKK signalsome maybe eluted with the specific MKP-1 peptide to which the antibody wasgenerated and fractionated further on a Mono Q column.

Throughout the fractionation, an in vitro kinase assay may be used tomonitor the IκB kinase activity of the IKK signalsome. In such an assay,the ability of a fraction to phosphorylate an appropriate substrate(such as IκBα (SEQ ID NO:1) or a derivative or variant thereof) isevaluated by any of a variety of means that will be apparent to those ofordinary skill in the art. For example, a substrate may be combined witha chromatographic fraction in a protein kinase buffer containing ³²Pγ-ATP, phosphatase inhibitors and protease inhibitors. The mixture maybe incubated for 30 minutes at 30° C. The reaction may then be stoppedby the addition of SDS sample buffer and analyzed using SDS-PAGE withsubsequent autoradiography. Suitable substrates include full length IκBα(SEQ ID NO: 1), polypeptides comprising the N-terminal 54 amino acids ofIκBα, full length IκBβ (SEQ ID NO:2) and polypeptides comprising theN-terminal 44 amino acids of IκBβ. Any of these substrates may be usedwith or without an N-terminal tag. One suitable substrate is a proteincontaining residues 1-54 of IκBα and an N-terminal GST tag (referred toherein as GST-IκBα 1-54; SEQ ID NO:3). To evaluate the specificity of anIκB kinase complex, IκBα mutants containing threonine or alanineresidues at positions 32 and 36, and/or other modifications, may beemployed.

Alternatively, an IKK signalsome may be prepared from its componentswhich are also encompassed by the present invention. Such components maybe produced using well known recombinant techniques, as described ingreater detail below. Components of an IKK signalsome may be native, ormay be variants of a native component (i.e., a component sequence maydiffer from the native sequence in one or more substitutions and/ormodifications, provided that the ability of a complex comprising thecomponent variant to specifically phosphorylate IκBα is notsubstantially diminished). Substitutions and/or modifications maygenerally be made in non-critical and/or critical regions of the nativeprotein. Variants may generally comprise residues of L-amino acids,D-amino acids, or any combination thereof. Amino acids may benaturally-occuring or may be non-natural, provided that at least oneamino group and at least one carboxyl group are present in the molecule;α- and β-amino acids are generally preferred. A variant may also containone or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Component variants may also, or alternatively, contain othermodifications, including the deletion or addition of amino acids thathave minimal influence on the activity of the polypeptide. Inparticular, variants may contain additional amino acid sequences at theamino and/or carboxy termini. Such sequences may be used, for example,to facilitate purification or detection of the component polypeptide. Ingeneral, the effect of one or more substitutions and/or modificationsmay be evaluated using the representative assays provided herein.

A component may generally be prepared from a DNA sequence that encodesthe component using well known recombinant methods. DNA sequencesencoding components of an IKK signalsome may be isolated by, forexample, screening a suitable expression library (i.e., a libraryprepared from a cell line or tissue that expresses IKK signalsome, suchas spleen, leukocytes, HeLa cells or Jurkat cells) with antibodiesraised against IKK signalsome or against one or more components thereof.Protein components may then be prepared by expression of the identifiedDNA sequences, using well known recombinant techniques.

Alternatively, partial sequences of the components may be obtained usingstandard biochemical purification and microsequencing techniques. Forexample, purified complex as described above may be run on an SDS-PAGEgel and individual bands may be isolated and subjected to proteinmicrosequencing. DNA sequences encoding components may then be preparedby amplification from a suitable human cDNA library, using polymerasechain reaction (PCR) and methods well known to those of ordinary skillin the art. For example, an adapter-ligated cDNA library prepared from acell line or tissue that expresses IKK signalsome (such as HeLa orJurkat cells) may be screened using a degenerate 5′ specific forwardprimer and an adapter-specific primer. Degenerate oligonucleotides mayalso be used to screen a cDNA library, using methods well known to thoseof ordinary skill in the art. In addition, known proteins may beidentified via Western blot analysis using specific antibodies.

Components of an IKK signalsome may also be identified by performing anyof a variety of protein-protein interaction assays known to those ofordinary skill in the art. For example a known component can be used as“bait” in standard two-hybrid screens to identify other regulatorymolecules, which may include IKK-1, IKK-2, NFκB1, RelA, IκBβ and/or p70S6 kinase (Kieran et al., Cell 62:1007-1018, 1990; Nolan et al., Cell64:961-69, 1991; Thompson et al., Cell 80:573-82, 1995; Grove et al.,Mol. Cell Biol. 11:5541-50, 1991).

Particularly preferred components of IKK signalsome are IκB kinases. AnIκB kinase may be identified based upon its ability to phosphorylate oneor more IκB proteins, which may be readily determined using therepresentative kinase assays described herein. In general, an IκB kinaseis incorporated into an IKK signalsome, as described herein, prior toperforming such assays, since an IκB kinase that is notcomplex-associated may not display the same phosphorylation activity ascomplex-associated IκB kinase. As noted above, an IκB kinase within anIKK signalsome specifically phosphorylates IκBα at residues S32 and S36,and phosphorylates IκBβ at residues 19 and 23, in response to specificstimuli.

As noted above, IKK-1 and IKK-2 are particularly preferred IκB kinases.IKK-1 and IKK-2 may be prepared by pooling the fractions from the Mono Qcolumn containing peak IκB kinase activity and subjecting the pooledfractions to preparative SDS gel electrophoresis. The intensity of twoprominent protein bands of ˜85 and ˜87 kDa (indicated by silver stain inFIG. 11B as IKK-1 and IKK-2 respectively) correlates with the profile ofIκB kinase activity. The ˜85 kDa band, corresponding to IKK-1, has beenidentified, within the context of the present invention, as CHUK(conserved helix-loop-helix ubiquitous kinase; see Connely and Marcu,Cell. Mol. Biol. Res. 41:537-49,1995). The ˜87 kDa band contains IKK-2.

Sequence analysis reveals that IKK-1 and IKK-2 are related proteinserine kinases (51% identity) containing protein interaction motifs(FIG. 13A). Both IKK-1 and IKK-2 contain the kinase domain at theN-terminus, and a leucine zipper motif and a helix-loop-helix motif intheir C-terminal regions. Northern analysis indicates that mRNAsencoding IKK-2 are widely distributed in human tissues, with transcriptsizes of ˜4.5 kb and 6 kb (FIG. 13B). The sequences of IKK-1 and IKK-2are also provided as SEQ ID NOs: 7 and 8, respectively.

It has been found, within the context of the present invention, thatrabbit reticulocyte lysate immunoprecipitates that contain IKK-1 orIKK-2 phosphorylate IκBα and IκBβ with the correct substrate specificity(see FIG. 14A). Altered versions of these kinases interfere withtranslocation of RelA to the nucleus of TNFα-stimulated HeLa cells.Accordingly, IKK-1 and IKK-2 appear to control a significant early stepof NFκB activation.

Other components of an IKK signalsome are also contemplated by thepresent invention. Such components may include, but are not limited to,upstream kinases such as MEKK-1 (Lee et al., Cell 88,:213-22, 1997;Hirano et al., J. Biol. Chem. 271:13234-38, 1996) or NIK (Malinin etal., Nature 385:540-44, 1997); adapter proteins that mediate anIKK-1:IKK-2 interaction; a component that crossreacts with anti-MKP-1;an inducible RelA kinase; and/or the E3 ubiquitin ligase that transfersmultiubiquitin chains to phosphorylated IκB (Hershko and Ciechanover,Annu. Rev. Biochem. 61:761-807, 1992).

A component of an IKK signalsome may generally be prepared from DNAencoding the component by expression of the DNA in cultured host cells,which may be stable cell lines or transiently transfected cells.Preferably, the host cells are bacteria, yeast, baculovirus-infectedinsect cells or mammalian cells. The recombinant DNA may be cloned intoany expression vector suitable for use within the host cell, usingtechniques well known to those of ordinary skill in the art. Anexpression vector may, but need not, include DNA encoding an epitope,such that the recombinant protein contains the epitope at the N- orC-terminus. Epitopes such as glutathione-S transferase protein (GST), HA(hemagglutinin)-tag, FLAG and Histidine-tag may be added usingtechniques well known to those of ordinary skill in the art.

The DNA sequences expressed in this manner may encode native componentsof an IKK signalsome, or may encode portions or variants of nativecomponents, as described above. DNA molecules encoding variants maygenerally be prepared using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis. Sections of the DNAsequence may also, or alternatively, be removed to permit preparation oftruncated polypeptides and DNA encoding additional sequences such as“tags” may be added to the 5′ or 3′ end of the DNA molecule.

IKK signalsome components may generally be used to reconstitute IKKsignalsome. Such reconstitution may be achieved in vitro by combiningIKK signalsome components in a suitable buffer. Alternatively,reconstitution may be achieved in vivo by expressing components in asuitable host cell, such as HeLa or HUVEC, as described herein.

Expressed IKK signalsome, or a component thereof, may be isolated insubstantially pure form. Preferably, IKK signalsome or a component isisolated to a purity of at least 80% by weight, more preferably to apurity of at least 95% by weight, and most preferably to a purity of atleast 99% by weight. In general, such purification may be achievedusing, for example, the representative purification methods describedherein or the standard techniques of ammonium sulfate fractionation,SDS-PAGE electrophoresis, and affinity chromatography. IKK signalsomeand components for use in the methods of the present invention may benative, purified or recombinant.

In one aspect of the present invention, an IKK signalsome and/or one ormore components thereof may be used to identify modulating agents, whichmay be antibodies (e.g., monoclonal), polynucleotides or other drugs,that inhibit or stimulate signal transduction via the NF-κB cascade.Modulation includes the suppression or enhancement of NF-κB activity.Modulation may also include suppression or enhancement of IκBphosphorylation or the stimulation or inhibition of the ability ofactivated (i.e., phosphorylated) IKK signalsome to phosphorylate asubstrate. Compositions that inhibit NF-κB activity by inhibiting IκBphosphorylation may include one or more agents that inhibit or blockIκBα kinase activity, such as an antibody that neutralizes IKKsignalsome, a competing peptide that represents the substrate bindingdomain of IκB kinase or a phosphorylation motif of IκB, an antisensepolynucleotide or ribozyme that interferes with transcription and/ortranslation of IκB kinase, a molecule that inactivates IKK signalsome bybinding to the complex, a molecule that binds to IκBα and preventsphosphorylation by IKK signalsome or a molecule that prevents transferof phosphate groups from the kinase to the substrate. Within certainembodiments, a modulating agent inhibits or enhances the expression oractivity of IKK-1 and/or IKK-2.

In general, modulating agents may be identified by combining a testcompound with an IKK signalsome, IκB kinase or a polynucleotide encodingan IκB kinase in vitro or in vivo, and evaluating the effect of the testcompound on the IκB kinase activity using, for example, a representativeassay described herein. An increase or decrease in kinase activity canbe measured by adding a radioactive compound, such as ³²P-ATP andobserving radioactive incorporation into a suitable substrate for IKKsignalsome, thereby determining whether the compound inhibits orstimulates kinase activity. Briefly, a candidate agent may be includedin a reaction mixture containing compounds necessary for the kinasereaction (as described herein) and IκB substrate, along with IKKsignalsome, IκB kinase or a polynucleotide encoding an IκB kinase. Ingeneral, a suitable amount of antibody or other agent for use in such anassay ranges from about 0.01 μM to about 10 μM. The effect of the agenton IκB kinase activity may then be evaluated by quantitating theincorporation of [³²P]phosphate into an IκB such as IκBα (or aderivative or variant thereof), and comparing the level of incorporationwith that achieved using IκB kinase without the addition of a candidateagent. Alternatively, the effect of a candidate modulating agent ontranscription of an IκB kinase may be measured, for example, by Northernblot analysis or a promoter/reporter-based whole cell assay.

Alternatively, for assays in which the test compound is combined with anIKK signalsome, the effect on a different IKK signalsome activity may beassayed. For example, an IKK signalsome also displays p65 kinaseactivity and IKK phosphatase activity. Assays to evaluate the effect ofa test compound on such activities may be performed using well knowntechniques. For example, assays for p65 kinase activity may generally beperformed as described by Zhong et al., Cell 89:413-24, 1997. Forphosphatase activity, an assay may generally be performed as describedby Sullivan et al., J. Biomolecular Screening 2:19-24, 1997, using arecombinant phosphorylated IκB kinase as a substrate.

In another aspect of the present invention, IKK signalsome or IκB kinasemay be used for phosphorylating an IκB such as IκBα (or a derivative orvariant thereof) so as to render it a target for ubiquitination andsubsequent degradation. IκB may be phosphorylated in vitro by incubatingIKK signalsome or IκB kinase with IκB in a suitable buffer for 30minutes at 30° C. In general, about 0.01 μg to about 9 μg of IκB kinasecomplex is sufficient to phosphorylate from about 0.5 μg to about 2 μgof IκB. Phosphorylated substrate may then be purified by binding toGSH-sepharose and washing. The extent of substrate phosphorylation maygenerally be monitored by adding [γ-³²P]ATP to a test aliquot, andevaluating the level of substrate phosphorylation as described herein.

An IKK signalsome, component thereof, modulating agent and/orpolynucleotide encoding a component and/or modulating agent may also beused to modulate NF-κB activity in a patient. Such modulation may occurby any of a variety of mechanisms including, but not limited to, directinhibition or enhancement of IκB phosphorylation using a component ormodulating agent; or inhibiting upstream activators, such as NIK or MEK,with IKK signalsome or a component thereof. As used herein, a “patient”may be any mammal, including a human, and may be afflicted with adisease associated with IκB kinase activation and the NF-κB cascade, ormay be free of detectable disease. Accordingly, the treatment may be ofan existing disease or may be prophylactic. Diseases associated with theNF-κB cascade include inflammatory diseases, neurodegenerative diseases,autoimmune diseases, cancer and viral infection.

Treatment may include administration of an IKK signalsome, a componentthereof and/or an agent which modulates IκB kinase activity. Foradministration to a patient, one or more such compounds are generallyformulated as a pharmaceutical composition. A pharmaceutical compositionmay be a sterile aqueous or non-aqueous solution, suspension oremulsion, which additionally comprises a physiologically acceptablecarrier (i.e., a non-toxic material that does not interfere with theactivity of the active ingredient). Any suitable carrier known to thoseof ordinary skill in the art may be employed in the pharmaceuticalcompositions of the present invention. Representative carriers includephysiological saline solutions, gelatin, water, alcohols, natural orsynthetic oils, saccharide solutions, glycols, injectable organic esterssuch as ethyl oleate or a combination of such materials. Optionally, apharmaceutical composition may additionally contain preservatives and/orother additives such as, for example, antimicrobial agents,anti-oxidants, chelating agents and/or inert gases, and/or other activeingredients.

Alternatively, a pharmaceutical composition may comprise apolynucleotide encoding a component of an IKK signalsome and/or amodulating agent (such that the component and/or modulating agent isgenerated in situ) in combination with a physiologically acceptablecarrier. In such pharmaceutical compositions, the polynucleotide may bepresent within any of a variety of delivery systems known to those ofordinary skill in the art, including nucleic acid, bacterial and viralexpression systems, as well as colloidal dispersion systems, includingliposomes. Appropriate nucleic acid expression systems contain thenecessary polynucleotide sequences for expression in the patient (suchas a suitable promoter and terminating signal). DNA may also be “naked,”as described, for example, in Ulmer et al., Science 259:1745-49, 1993.

Various viral vectors that can be used to introduce a nucleic acidsequence into the targeted patient's cells include, but are not limitedto, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Preferably, the retroviralvector is a derivative of a murine or avian retrovirus including, butnot limited to, Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A retroviral vector may additionally transfer orincorporate a gene for a selectable marker (to aid in the identificationor selection of transduced cells) and/or a gene that encodes the ligandfor a receptor on a specific target cell (to render the vector targetspecific). For example, retroviral vectors can be made target specificby inserting a nucleotide sequence encoding a sugar, a glycolipid, or aprotein. Targeting may also be accomplished using an antibody, bymethods known to those of ordinary skill in the art.

Viral vectors are typically non-pathogenic (defective), replicationcompetent viruses, which require assistance in order to produceinfectious vector particles. This assistance can be provided, forexample, by using helper cell lines that contain plasmids that encodeall of the structural genes of the retrovirus under the control ofregulatory sequences within the LTR, but that are missing a nucleotidesequence which enables the packaging mechanism to recognize an RNAtranscript for encapsulation. Such helper cell lines include (but arenot limited to) Ψ2, PA317 and PA12. A retroviral vector introduced intosuch cells can be packaged and vector virion produced. The vectorvirions produced by this method can then be used to infect a tissue cellline, such as NIH 3T3 cells, to produce large quantities of chimericretroviral virions.

Another targeted delivery system for polynucleotides is a colloidaldispersion system. Colloidal dispersion systems include macromoleculecomplexes, nanocapsules, microspheres, beads, and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system for use as a delivery vehicle invitro and in vivo is a liposome (i.e., an artificial membrane vesicle).It has been shown that large unilamellar vesicles (LUV), which range insize from 0.2-4.0 μm can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci. 6:77, 1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques 6:882, 1988).

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity and may be, for example, organ-specific, cell-specific,and/or organelle-specific. Mechanistic targeting can be distinguishedbased upon whether it is passive or active. Passive targeting utilizesthe natural tendency of liposomes to distribute to cells of thereticuloendothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

Routes and frequency of administration, as well doses, will vary frompatient to patient. In general, the pharmaceutical compositions may beadministered intravenously, intraperitoneally, intramuscularly,subcutaneously, intracavity or transdermally. Between 1 and 6 doses maybe administered daily. A suitable dose is an amount that is sufficientto show improvement in the symptoms of a patient afflicted with adisease associated with the NF-κB cascade. Such improvement may bedetected by monitoring inflammatory responses (e.g., edema, transplantrejection, hypersensitivity) or through an improvement in clinicalsymptoms associated with the disease. The dosage may generally varydepending on the nature of the modulating agent and the disease to betreated. Typically, the amount of modulating agent present in a dose, orproduced in situ by DNA present in a dose, ranges from about 1 μg toabout 200 mg per kg of host. Suitable dose sizes will vary with the sizeof the patient, but will typically range from about 10 mL to about 500mL for 10-60 kg animal.

In another aspect, the present invention provides methods for detectingthe level of stimulus-inducible IκB kinase activity in a sample. Thelevel of IκB kinase activity may generally be determined via animmunokinase assay, in which IKK signalsome is first immunoprecipitatedwith an antibody that binds to the complex. The immunoprecipitatedmaterial is then subjected to a kinase assay as described herein.Substrate specificity may be further evaluated as described herein todistinguish the activity of a stimulus-inducible IκB kinase complex fromother kinase activities.

The present invention also provides methods for detecting the level ofIKK signalsome, or a component thereof, in a sample. The amount of IKKsignalsome, IκB kinase or nucleic acid encoding IκB kinase, maygenerally be determined using a reagent that binds to IκB kinase, or toDNA or RNA encoding a component thereof. To detect nucleic acid encodinga component, standard hybridization and/or PCR techniques may beemployed using a nucleic acid probe or a PCR primer. Suitable probes andprimers may be designed by those of ordinary skill in the art based onthe component sequence To detect IKK signalsome or a component thereof,the reagent is typically an antibody, which may be prepared as describedbelow.

There are a variety of assay formats known to those of ordinary skill inthe art for using an antibody to detect a protein in a sample. See,e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. For example, the antibody may be immobilized ona solid support such that it can bind to and remove the protein from thesample. The bound protein may then be detected using a second antibodythat binds to the antibody/protein complex and contains a detectablereporter group. Alternatively, a competitive assay may be utilized, inwhich protein that binds to the immobilized antibody is labeled with areporter group and allowed to bind to the immobilized antibody afterincubation of the antibody with the sample. The extent to whichcomponents of the sample inhibit the binding of the labeled protein tothe antibody is indicative of the level of protein within the sample.Suitable reporter groups for use in these methods include, but are notlimited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin.

Antibodies encompassed by the present invention may be polyclonal ormonoclonal, and may bind to IKK signalsome and/or one or more componentsthereof (e.g., IKK-1 and/or IKK-2). Preferred antibodies are thoseantibodies that inhibit or block IκB kinase activity in vivo and withinan in vitro assay, as described above. Other preferred antibodies arethose that bind to one or more IκB proteins. As noted above, antibodiesand other agents having a desired effect on IκB kinase activity may beadministered to a patient (either prophylactically or for treatment ofan existing disease) to modulate the phosphorylation of an IκB, such asIκBα, in vivo.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising the protein of interestis initially injected into a suitable animal (e.g., mice, rats, rabbits,sheep and goats), preferably according to a predetermined scheduleincorporating one or more booster immunizations, and the animals arebled periodically. Polyclonal antibodies specific for the protein maythen be purified from such antisera by, for example, affinitychromatography using the protein coupled to a suitable solid support.

Monoclonal antibodies specific for an IKK signalsome or a componentthereof may be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the complex and/or component of interest). Such celllines may be produced, for example, from spleen cells obtained from ananimal immunized as described above. The spleen cells are thenimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. For example,the spleen cells and myeloma cells may be combined with a nonionicdetergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

In a related aspect of the present invention, kits for detecting thelevel of IKK signalsome, IκB kinase, nucleic acid encoding IκB kinaseand/or IκB kinase activity in a sample are provided. Any of a variety ofsamples may be used in such assays, including eukaryotic cells,bacteria, viruses, extracts prepared from such organisms and fluidsfound within living organisms. In general, the kits of the presentinvention comprise one or more containers enclosing elements, such asreagents or buffers, to be used in the assay.

A kit for detecting the level of IKK signalsome, IκB kinase or nucleicacid encoding IκB kinase typically contains a reagent that binds to thecompound of interest. To detect nucleic acid encoding IκB kinase, thereagent may be a nucleic acid probe or a PCR primer. To detect IKKsignalsome or IκB kinase, the reagent is typically an antibody. Suchkits also contain a reporter group suitable for direct or indirectdetection of the reagent (i.e., the reporter group may be covalentlybound to the reagent or may be bound to a second molecule, such asProtein A, Protein G, immunoglobulin or lectin, which is itself capableof binding to the reagent). Suitable reporter groups include, but arenot limited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin. Such reporter groups may be used todirectly or indirectly detect binding of the reagent to a samplecomponent using standard methods known to those of ordinary skill in theart.

In yet another aspect, IKK signalsome may be used to identify one ormore native upstream kinases (i.e., kinases that phosphorylate andactivate IKK signalsome in vivo) or other regulatory molecules thataffect IκB kinase activity (such as phosphatases or molecules involvedin ubiquitination), using methods well known to those of ordinary skillin the art. For example, IKK signalsome components may be used in ayeast two-hybrid system to identify proteins that interact with suchcomponents. Alternatively, an expression library may be screened forcDNAs that phosphorylate IKK signalsome or a component thereof.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Recruitment of NFκB into IKK Sigmalsome DuringActivation

This example illustrates the recruitment of NFκB into a protein complex(the IKK signalsome) containing IκB kinase and other signaling proteins.

Cytoplasmic extracts of either unstimulated or stimulated Jurkat cellswere fractionated on a Superdex 200 gel filtration column, and IκBα wasvisualized by immunoblot analysis. Jurkat cells were grown to a celldensity of 1.5×10⁶ cells/ml and either not stimulated or induced for 10minutes with PMA (50 ng/ml)/PHA (1 μg/ml). Cells were harvested andresuspended in two volumes HLB buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄,1 mM benzamidine, 2 mM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 mM DTT), made 0.1% NP40 and left on ice for 15minutes, and lysed with a glass Dounce homogenizer. The nuclei werepelleted at 10,000 rpm for 20 minutes in a Sorval SS34 rotor. Thesupernatant was further centrifuged at 40,000 rpm for 60 min in a Ti50.1rotor. All procedures were carried out at 4° C. The S-100 fraction wasconcentrated and chromatographed on Hi Load 16/60 Superdex 200 prepgrade gel filtration column that was equilibrated in GF buffer (20 mMTris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 0.025%Brij 35, 1 mM benzamidine, 2 mM PMSF, 10 mM β-glycerophosphate, 10 mMNaF, 10 mM PNPP, 300 μM Na₃VO₄, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 mM DTT). Isolated fractions were analyzed by westernblot analysis using either anti-IκBα or anti-JNK antibodies (Santa Cruz,Inc., Santa Cruz, Calif.).

As shown in FIG. 1A, IκBα in cell extracts from unstimulated cellseluted with an apparent molecular weight of ˜300 kDa (lanes 5-7),consistent with the chromatographic properties of the inactive NFκB-IκBcomplex (Baeuerle and Baltimore, Genes Dev. 3:1689-98, 1989). Incontrast, phosphorylated IκBα (from cells stimulated for periods tooshort to permit complete degradation of the protein) migrated at ˜600kDa on the same chromatography columns (lanes 2, 3). This difference inmigration was specific for IκB, since analysis of the same fractionsindicated that the Jun N-terminal kinases JNK1 and JNK2 migrated withlow apparent molecular weight and showed no difference inchromatographic behavior between stimulated and unstimulated cells.Stimulation-dependent recruitment of IκB into this larger proteincomplex corresponded with the appearance of phosphorylated IκB,suggesting that the complex contained the specific IκB kinases thatmediate IκB phosphorylation. These results demonstrate that that NFκBactivation involves recruitment into a protein complex (the IKKsignalsome) containing IκB kinase and other signaling proteins.

Example 2 Partial Purification of IKK Signalsome and Identification ofCo-Purifying Components

This Example illustrates the fractionation of extracts containing IκBkinase. Whole cell extracts from TNFα-stimulated cells were fractionatedby gel filtration, ion exchange, and other chromatographic methods, asdescribed above. IκB kinase activity in the fractions was assayed byphosphorylation of GST-IκBα (1-54) (SEQ ID NO:3) or GST-IκB β (1-44)(SEQ ID NO:4). Kinase assays were performed in 20 mM HEPES pH 7.7, 2 MMMgCl₂, 2 mM MnCl₂, 10 μM ATP, 1-3 μCi γ-[³²P]-ATP, 10 mMβ-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1 mMbenzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/mlpepstatin, 1 mM DTT) at 30° C. for 30 to 60 minutes in the presence ofthe indicated substrate. The kinase reaction was stopped by the additionof 6×SDS-PAGE sample buffer, subjected to SDS-PAGE analysis andvisualized using autoradiography. GST-IκB substrates for use in theabove assay were prepared using standard techniques for bacteriallyexpressed GST-protein (see Current Protocols in Molecular Biology2:16.7.1-16.7.7, 1996). Bacterial cells were lysed, GST proteins werepurified via binding to GST-agarose beads, washed several times, elutedfrom the beads with glutathione, dialyzed against kinase assay bufferand stored at −80° C. The specificity of the kinase was established byusing mutant GST-IκBα (1-54) in which serines 32, 36 had been mutated tothreonine (SEQ ID NO:5), and GST-IκBβ (1-44) in which serines 19, 23 hadbeen mutated to alanine (SEQ ID NO:6).

IκB kinase activity was not observed in extracts from unstimulatedcells, while stimulation with TNFα for 5-7 minutes resulted in stronginduction of kinase activity. As shown in FIG. 1B, the IκB kinaseactivity from stimulated cells chromatographed on gel filtration as abroad peak of ˜500-700 kDa, consistent with its presence in a largeprotein complex potentially containing other components required forNFκB activation.

NFκB activation is known to occur under conditions that also stimulateMAP kinase pathways (Lee et al., Cell 88:213-22, 1997; Hirano, et al.,J. Biol. Chem. 271:13234-38, 1996). Accordingly, further experimentswere performed to detect proteins associated with MAP kinase andphosphatase cascades at various stages of purification of the IKKsignalsome. In addition to RelA and IκBβ, MEKK-1 and twotyrosine-phosphorylated proteins of ˜55 and ˜40 kDa copurified with IκBkinase activity (FIG. 1C). Antibodies to Rel A and IκBβ were obtainedfrom Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), and antibodiesto MEKK-1 were obtained from Upstate Biotechnology (Lake Placid, N.Y.).Other signaling components, including PKCζ, PP1 and PP2A, were detectedin the same fractions as the IκB kinase in early chromatographic stepsbut did not copurify at later chromatographic steps (data not shown).Most interestingly, an unidentified protein of ˜50 kDa, detected by itscrossreaction with an antibody to MKP-1, copurified with IκB kinasethrough several purification steps (FIG. 1C). This protein is unlikelyto be MKP-1 itself, since the molecular weight of authentic MKP-1 is 38kDa.

Example 3 Preparation of IKK Signalsome from HeLa S3 Cell Extracts

This Example illustrates an alternate preparation of an IKK signalsome,and the characterization of the complex.

HeLa S3 cells were grown to a cell density of approximately 0.6×10⁶/mL,concentrated 10 fold and induced with TNFα (30 ng/mL) for seven minutes.Two volumes of ice-cold PBS containing phosphatase inhibitors (10 mMsodium fluoride, 0.3 mM sodium orthovanadate and 20 mMβ-glycerophosphate) were then added. The cells were spun down, washedonce with ice-cold PBS containing phosphatase inhibitors and snapfrozen.

Standard protocols were then used to prepare cytoplasmic and nuclearextracts. More specifically, the frozen HeLa S3 cell pellet wasquick-thawed at 37° C., resuspended in 2 volumes of ice-cold HypotonicLysis Buffer (20 mM Tris pH 8.0, 2 mM EDTA, 1 mM EGTA, 10 mMβ-glycerophosphate, 10 mM NaF, 10 mM PNPP, 0.3 mM Na₂VO₄, 5 mM sodiumpyrophosphate, 1 mM benzamidine, 2 mM PMSF, 10 μg/mL aprotinin, 1 μg/mLleupeptin and 1 μg/mL pepstatin), and left to incubate on ice for 30min. The swollen cells were then dounced 30 times using a tight pestleand the nuclei were pelleted at 10,000 rpm for 15 minutes at 4° C. Thesupernatant was clarified via ultracentrifugation (50,000 rpm for 1 hourat 4° C.) to generate the final cytoplasmic extract. Thenuclear/membrane pellet was resuspended in an equal volume of High SaltExtraction Buffer (20 mM Tris pH 8.0, 0.5M NaCl, 1 mM EDTA, 1 mM EGTA,0.25% Triton X-100, 20 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 0.3mM Na₂VO₄, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, 10 μg/mL aprotinin, 1μg/mL leupeptin and 1 μg/mL pepstatin) and allowed to rotate at 4° C.for 30 minutes. Cell debris was removed via centrifugation at 12,500 rpmfor 30 minutes at 4° C. and the resulting supernatant was saved as thenuclear/membrane extract.

These extracts were then independently subjected to a series ofchromatographic steps (shown in FIG. 2) using a Pharmacia FPLC system(Pharmacia Biotech, Piscataway, N.J.):

-   -   (1) Q Sepharose (Pharmacia Biotech, Piscataway, N.J.)—the column        was run with a linear gradient starting with 0.0M NaCl Q Buffer        (20 mM Tris pH 8.0, 0.5 mM EDTA, 0.5 mM EGTA, 0.025% Brij 35, 20        mM β-glycerophosphate, 10 mM NaF, 0.3 mM Na₂VO₄, 1 mM        benzamidine, 1 mM PMSF, 2 mM DTT, 10 μg/mL aprotinin, 1 μg/mL        leupeptin and 1 μg/mL pepstatin) and ending with 0.5M NaCl Q        Buffer. The IκBα kinase activity eluted between 0.25 and 0.4 M        NaCl.    -   (2) Gel Filtration HiLoad 16/60 Superdex 200) (Pharmacia        Biotech, Piscataway, N.J.)—the column was run with Gel        Filtration Buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 1        mM EGTA, 0.05% Brij 35, 20 mM β-glycerophosphate, 10 mM NaF, 0.3        mM Na₂VO₄, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, 10 μg/mL        aprotinin, 1 μg/mL leupeptin and 1 μg/m L pepstatin). The peak        IκBα kinase activity eluted at 40-48 mL, which corresponds to a        molecular weight of 731 kD to 623 kD.    -   (3) HR 5/5 Mono Q (Pharmacia Biotech, Piscataway, N.J.)—the        column was run with a linear gradient starting with 0.0M NaCl Q        Buffer and ending with 0.5M NaCl Q Buffer (without Brij        detergent to prepare sample for Phenyl Superose column). The        IκBα kinase activity eluted between 0.25 and 0.4 M NaCl.    -   (4) HR Phenyl Superose (Pharmacia Biotech, Piscataway, N.J.)—the        column was run with a linear gradient of 1.0M to 0.0M ammonium        sulfate in Phenyl Superose Buffer (20 mM Tris pH 8.0, 0.25 mM        EDTA, 1 mM EGTA, 20 mM β-glycerophosphate, 10 mM NaF, 0.1 mM        Na₂VO₄, 1 mM benzamidine, 1 mM PMSF, 1 mM DTT, 10 μg/mL        aprotinin, 1 μg/mL leupeptin and 1 μg/mL pepstatin). The IκBα        kinase activity eluted between 0.35 and 0.2 M ammonium sulfate.    -   (5) Gel Filtration Superdex 200 HR 10/30 (Pharmacia Biotech,        Piscataway, N.J.)—the column was run with Gel Filtration Buffer        (see (2), above). The peak of activity eluted at 8-10 mL, which        corresponds to a molecular weight of 720 kD to 600 kD.    -   (6) HR 5/5 Mono Q—the column was run as in (3) above except that        the 0.05% Brij 35 was included in all Q buffers.

IκBα kinase activity, with similar substrate specificity and molecularweight, was isolated from both the cytoplasmic and nuclear/membraneextracts.

At each step of the fractionation, IκB kinase activity was monitoredusing an in vitro assay. The assay was performed by combining 2 μg ofthe respective IκB substrate (GST-IκBα 1-54 (wildtype) or GST-IκBα(S32/36 to T), as described below) with 3-5 μL chromatographic fractionand 20 μL of Kinase Assay Buffer (20 mM HEPES pH 7.4, 10 mM MgCl₂, 10 mMMnCl₂, 20 mM NaCl, 1 mM DTT, 20 mM PNPP, 20 μM ATP, 20 mMα-glycerophosphate, 10 mM NaF, 0.1 mM Na₂VO₄, 1 mM benzamidine, 1 mMPMSF) containing γ³²P-ATP, and incubating for 30 minutes at 30° C. Thekinase reaction was terminated by adding 8 μL of 6×SDS-PAGE samplebuffer. The entire sample was run on a 12% polyacrylamide gel, dried andsubjected to autoradiography.

IκB substrates for use in the above assay were prepared using standardtechniques (see Haskill et al., Cell 65:1281-1289, 1991). The GST-IκBα1-54 (wildtype) or GST-IκBα (S32/36 to T) substrates were prepared usingstandard techniques for bacterially expressed GST-protein. Bacterialcells were lysed, GST proteins were purified via binding to GST-agarosebeads, washed several times, eluted from the beads with glutathione,dialyzed against 50 mM NaCl Kinase Assay Buffer and stored at −80° C.

The TNFα-inducibility of IκB kinase activity was initially evaluated byWestern blot analysis of the levels of IκB in HeLa S3 cytoplasmicextracts following gel filtration. IκBα was assayed by running 18 μL ofthe gel filtration fractions on 10% SDS PAGE, transferring toNitrocellulose Membrane (Hybond-ECL, Amersham Life Sciences, ArlingtonHeight, Ill.) using standard blotting techniques and probing withanti-IκBα antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.). TNFα-inducibility was evaluated by comparing the level of IκBαin cells that were (FIG. 3B) and were not (FIG. 3A) exposed to TNFα (30ng/mL for seven minutes, as described above).

The IκB kinase activity of these cytoplasmic extracts was evaluatedusing the kinase assay described above. As shown in FIG. 4B, the extractof TNFα-treated cells phosphorylated GST-IκBα 1-54 (wildtype), while theuntreated cell extract showed significantly lower levels of IκBα kinaseactivity (FIG. 4A).

Cytoplasmic extracts of TNFα-treated HeLa S3 cells (following QSepharose fractionation) were also subjected to in vitro kinase assays,using the N-terminal portion of IκBα (residues 1 to 54) as substrate.With the wild type substrate, phosphorylation of GST-IκBα 1-54 wasreadily apparent (FIG. 5A). In contrast, substrate containing threoninesubstitutions at positions 32 and 36 was not phosphorylated (FIG. 5B).

Following chromatographic fractionation by Q Sepharose, Superdex 200,MonoQ Sepharose and Phenyl Superose, in vitro kinase assay showedsubstantial purification of the IκB kinase activity (FIG. 6A). Furtherpurification of the IκB kinase was achieved by passing the sample over,in series, an analytical Superdex 200 and Mono Q HR 5/5, resulting in 8major protein bands as determined by silver staining. As before, the useof substrate containing threonine substitutions at positions 32 and 36markedly reduced the phosphorylation (FIG. 6B). These resultsdemonstrate the purification of a stimulus-inducible IκBα kinasecomplex, which specifically phosphorylates serine residues at positions32 and 36 of IκBα without the addition of exogenous factors.

Example 4 Immunoprecipitation of IKK Signalsome Using Anti MKP-1Antibodies

This Example illustrates the immunoprecipitation of IκB kinase activityfrom cytoplasmic extracts prepared from stimulated cells.

A. Immunoprecipitation of IκB Kinase Complex from HeLa Cells

HeLa cells were TNF-α-treated (30 μg/mL, 7 minutes) and fractionated bygel filtration as described in Example 3. Twenty μL of gel filtrationfraction #6 (corresponding to about 700 kD molecular weight) and 1 μgpurified antibodies raised against MKP-1 (Santa Cruz Biotechnology,Inc., Santa Cruz, Calif.) were added to 400 μL of ice cold 1×Pull DownBuffer (20 mM Tris pH 8.0,250 mM NaCl, 0.05% NP-40, 3 mM EGTA, 5 mMEDTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1mM benzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1μg/ml pepstatin, 1 mM DTT). The sample was gently rotated for 1 hour at4° C., at which time 40 μL of protein A-agarose beads (50:50 slurry,Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) was added. Thesample was then rotated for an additional 1.5 hours at 4° C. The proteinA-agarose beads were pelleted at 3,000 rpm for 2 minutes at 4° C. andthe pellet was washed three times with ice cold Pull Down Buffer (800 μLper wash).

The pellet was subjected to the standard in vitro IκBα kinase assay (asdescribed above) using either 2 μg GST-IκBα1-54 (wildtype) or 2μg-GST-IκBα1-54 (S32/36 to T) as the substrate.

The results, shown in FIG. 7, demonstrate that antibodies directedagainst MKP-1 immunoprecipitate the stimulus-inducible IκBα kinaseactivity. The substrate specificity of this IκKα kinase activitycorresponds to what has been described in vivo (strong phosphorylationof the GST-IκBα 1-54 (wildtype) and no phosphorylation usingGST-IκBα1-54 (S32/36 to T).

B. Characterization of Immunoprecipitated IKK Signalsome

For these studies, small scale immunoprecipitation were performed usingtwo 150 mm plates of HeLa cells (one stimulated and one unstimulated).Whole cell lysates were diluted 4-fold with 2×Pull-Down Buffer (40 mMTris pH 8.0, 500 mM NaCl, 0.1% NP-40, 6 mM EDTA, 6 mM EGTA, 10 mMβ-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1 mMbenzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/mlpepstatin, 1 mM DTT) and 2-4 μg of the indicated antibody was added.Lysates were incubated on ice for 1-2 hours, 10 μl of Protein A or Gbeads were added, and lysates were left to incubate with gentle rotationfor an additional 1 hour at 4° C. The immunoprecipitate was then washed3 times with 2×Pull-Down Buffer, 1×with kinase buffer without ATP andsubjected to a kinase assay as described in Example 2. There was nonoticeable loss in IκB kinase activity when the immunoprecipitate wassubjected to more rigorous washing, such as in RIPA buffer (20 mM Tris,250 mM NaCl, 1% NP-40, 1% DOC, 0.1% SDS, 3 mM EDTA, 3 mM EGTA. 10 mM,β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 μM Na₃VO₄, 1 mMbenzamidine, 2 μM PMSF, 10 μg/ml aprotonin, 1 μg/ml leupeptin, 1 μg/mlpepstatin, 1 mM DTT) or washes up to 3.5 M urea.

Of a large panel of antibodies tested, one of three anti-MKP-1antibodies efficiently co-immunoprecipitated an inducible IκB kinaseactivity from HeLa cells as well as primary human umbilical veinendothelial cells (HUVEC). The co-immunoprecipitated kinase (IKKsignalsome kinase) was inactive in unstimulated HeLa cells, but wasrapidly activated within minutes of TNFα stimulation (FIG. 8A, toppanel). The IKK signalsome kinase did not phosphorylate a mutantGST-IκBα protein in which serine residues 32 and 36 had been mutated tothreonine (FIG. 8A top panel, even-numbered lanes). Activation of thesignalsome kinase was maximal at 5 minutes and declined thereafter, atime course consistent with the time course of IκBα phosphorylation anddegradation under the same conditions (FIG. 8A, bottom panel). Asexpected, the signalsome IκB kinase was also activated by stimulation ofcells with IL-1 or PMA (FIG. 8B, lanes 1-4); moreover, its activity wasinhibited in cells treated with TPCK, a known inhibitor of NFκBactivation (FIG. 8B, lane 7). Additionally, the IKK signalsome kinasespecifically phosphorylated full-length wild-type IκBα, but not a mutantIκBα bearing the serine 32, 36 to alanine mutations, in the context of aphysiological RelA-IκBα complex (FIG. 8C, lanes 3, 4). Together theseresults indicate that the anti-MKP-1 antibody co-immunoprecipitated theIKK signalsome. The signalsome-associated IκB kinase met all thecriteria expected of the authentic IκB kinase and had no detectable IκBαC-terminal kinase activity.

The specificity of the IKK signalsome kinase was further established bykinetic analysis and by examining its activity on various peptides andrecombinant protein substrates (FIG. 9A). For these studies, syntheticpeptides (Alpha Diagnostics International, San Antonio, Tex.) wereprepared with the following sequences:

-   -   IκBα (21-41): CKKERLLDDRHDSGLDSMKDEE (SEQ ID NO:11)    -   IκBα (21-41) S/T mutant: CKKERLLDDRHDTGLDTMKDEE (SEQ ID NO:12)    -   c-Fos(222-241): DLTGGPEVAT(PO3)PESEEAFLP (SEQ ID NO:13)    -   MKP-1: CPTNSALNYLKSPITTSPS (SEQ ID NO:14)    -   cJun (56-70): CNSDLLTSPDVGLLK (SEQ ID NO:15)    -   cJun (65-79): CVGLLKLASPELERL (SEQ ID NO:16)

Phosphorylation of these peptides (100 μM) was performed using a kinasereaction as described above. Reactions were for one hour at roomtemperature and were terminated by the addition of SDS-PAGE loadingbuffer. SDS-PAGE with a 16% Tris/tricine gel (Novex, San Diego, Calif.)or a 4-20% Tris/glycine gel (Novex, San Diego, Calif.) was used tocharacterize the reaction products. Gels were washed, dried in vacuo,and exposed to autoradiographic film.

Inhibition of immunopurified IKK signalsome activity was measured by ³²Pincorporation into GST-IκKα (1-54) in a discontinuous assay using thereaction conditions described above. The concentrations of GST-IκBα(1-54) and ATP used in the inhibition studies were 0.56 μM and 3 μM,respectively. Enzymatic reactions (32 μL) were carried out in wells of a96 well assay plate for one hour at room temperature and terminated withthe addition of trichloroacetic acid (TCA) (150 μL/well of 12.5% w/v).The subsequent 20 minute incubation with TCA precipitated the proteinsbut not peptides from solution. The TCA precipitate was collected on96-well glass fiber plates (Packard) and washed 10 times withapproximately 0.3 mL per well of Dulbecco's phosphate buffered saline pH7.4 (Sigma) using a Packard Filtermate 196. Scintillation fluid (0.50mL, MicroScint, Packard) was added to each well and the plate wasanalyzed for ³²P using a Packard TopCount scintillation counter. Lessthan 10% of ATP was turned over in the course of the assay reaction,ensuring that the kinetic data represented initial rate data. Theinhibition constant of the P32, 36 peptide was determined by Dixonanalysis (Dixon and Webb, In Enzymes (Academic Press: New York, ed. 3,1979), pp. 350-51.

The kinase displayed normal Michaelis-Menten kinetics, suggesting thatit was not a mixture of diverse unrelated kinases. The kinase wascapable of phosphorylating an IκBα (21-41) peptide (FIGS. 9A and 9B)) aswell as two different IκBα (21-41) peptides, each bearing a free serineat either position 32 or 36 and phosphoserine at the other position(FIGS. 9A and 9B, lanes 2, 3). As expected, a peptide withphosphoserines at both positions was not phosphorylated at all (FIG. 9B,top), indicating that there was no significant turnover of thephosphates under our reaction conditions. These experiments indicatedthat both serines 32 and 36 were phosphoacceptor sites for the IKKsignalsome kinase, thus distinguishing it from other kinases such aspp90Rsk which phosphorylates IκBα only at serine 32 (Schouten, et al.,EMBO J. 16:3133-44, 1997).

Although the IKK signalsome kinase efficiently phosphorylated IκBpeptides, it did not phosphorylate the c-Fos phosphopeptide containing afree serine and a free threonine (FIG. 9B, top), two c-Jun peptidescontaining serine 63 and 73, respectively, (FIG. 9A, top panel, lanes 4,5), or an MKP-1 peptide containing four serines and three threonines(FIG. 9A, lane 6). The latter peptides were substrates for JNK2 (FIG.9A, bottom panel, lanes 4-6). An IκBα (21-41) peptide in which serines32 and 36 were replaced by threonines was phosphorylated by thesignalsome at least 10-fold less well than the wild-typeserine-containing peptide, consistent with the slower phosphorylationand degradation kinetics of IκBα (S32/36 to T) in cells (DiDonato etal., Mol Cell. Biol. 16:1295-1304, 1996), and the preference of thekinase for serine over threonine at positions 32, 36 in both full-lengthIκBα and GST-IκBα (1-54) (FIGS. 8A and C). In addition, the kinasephosphorylated GST-IκBβ (1-54), albeit with lower affinity. In mostexperiments, IκB kinase activity was also associated with strong RelAkinase activity (FIG. 8C, lanes 3, 4), but no activity was observedtowards several other substrates including myelin basic protein (MBP),GST-ATF2 (1-112), GST-cJun (1-79), GST-ERK3, GST-Elk (307428), GST-p38,and a GST fusion protein containing the C-terminal region of IκBα(242-314).

The specificity of the IKK signalsome kinase was further emphasized byits susceptibility to product inhibition (FIG. 9B, bottom). The kinasewas strongly inhibited by a doubly-phosphorylated IκBα peptide bearingphosphoserines at both positions 32 and 36, but not by the unrelatedc-Fos phosphopeptide that contained a single phosphothreonine. Thesingly-phosphorylated and the unphosphorylated IκBα peptides were lesseffective inhibitors.

Example 5 Absence of Free Ubiguitin in Purified IKK Signalsome

This example illustrates the absence of detectable free ubiquitin with aIKK signalsome prepared as in Example 3. Standard western blotprocedures were performed (Amersham Life Science protocol, ArlingtonHeights, Ill.). 100 ng ubiquitin, 10 ng ubiquitin and 20 ul purifiedIκBα kinase complex was subjected to 16% Tricine SDS-PAGE (Novex, SanDiego, Calif.), transferred to Hybond ECL Nitrocellulose membrane(Amersham Life Science, Arlington Heights, Ill.), and probed withantibodies directed against ubiquitin (MAB1510; Chemicon, Temecula,Calif.). The results are shown in FIG. 10. Free ubiquitin could not bedetected in the purified IκBα kinase preparation (even at very longexposures). The complexes described herein do not require addition ofendogenous ubiquitin to detect IκBα kinase activity, nor is freeubiquitin a component in the purified IκBα kinase preparations of thepresent invention.

Example 6 Purification of the NFκB Signalsome and Identification ofIKK-1 and IKK-2

This Example illustrates a two-step affinity method for purification ofthe IKK signalsome, based on its recognition by the MKP-1 antibody(depicted in FIG. 11A) and the identification of IκB kinases.

For large scale IKK signalsome purification, HeLa S3 cells werestimulated for 7 minutes with 20 ng/ml TNFα (R&D Systems, Minneapolis,Minn.), harvested, whole cell lysates were prepared (1.2 g totalprotein) and approximately 5 mg of anti-MKP-1 antibody (Santa CruzBiotechnology, Inc., Santa Cruz, Calif.) was added and incubated at 4°C. for 2 hours with gentle rotation. Subsequently, 50 ml of Protein Aagarose (Calbiochem, San Diego, Calif.) was added and the mixture wasincubated for an additional 2 hours. The immunoprecipitate was thensequentially washed 4×Pull-Down Buffer, 2×RIPA buffer, 2×Pull-DownBuffer, 1×3.5 M urea-Pull-Down Buffer and 3×Pull-Down Buffer. Theimmunoprecipitate was then made into a thick slurry by the addition of10 ml of Pull-Down Buffer, 25 mg of the specific MKP-1 peptide to whichthe antibody was generated (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.) was added, and the mixture was incubated overnight at 4° C. withgentle rotation. The eluted IKK signalsome was then desalted on PD 10desalting columns (Pharmacia Biotech, Piscataway, N.J.) equilibratedwith 50 mM Q buffer and chromatographed on a Mono Q column (PharmaciaBiotech, Piscataway, N.J.). Fractions containing peak IκB kinaseactivity were pooled, concentrated and subjected to preparativeSDS-PAGE. The intensity of two prominent protein bands of ˜85 and ˜87kDa (indicated by silver stain in FIG. 11B as IKK-1 and IKK-2respectively) correlated with the profile of IκB kinase activity.

Coomassie stained ˜85 and ˜87 kDa bands were excised, in-gel digestedwith trypsin (Wilm et al., Nature 37:466-69, 1996) and a small aliquotof the supernatant was analyzed by high mass accuracy MALDI peptide massmapping, as described by Shevchenko et al., Proc. Natl. Acad. Sci. USA93:14440-45, 1996. The peptide mass map from the IKK-1 band was searchedagainst a comprehensive protein sequence database using the programPeptideSearch developed in house at EMBL Heidelberg. Eight measuredpeptide masses matched calculated tryptic peptide masses from CHUK(conserved helix-loop-helix ubiquitous kinase; Connely and Marcu, Cell.Mol. Biol. Res. 41:537-49, 1995) within 30 ppm, unambiguouslyidentifying the protein. The peptide mass map of the IKK-2 band did notresult in a clear identification and therefore the sample was subjectedto nanoelectrospray mass spectrometry (Wilm and Mann, Anal Chem. 68:1-8,1996). The peptide mixture obtained after extraction of the gel piecewas micropurified on a capillary containing 50 nL of POROS R2 resin(PerSeptive Biosystems, Framingham, Mass.). After washing, the peptideswere step-eluted with 0.5 μl of 50% MeOH in 5% formic acid into ananoelectrospray needle. This needle was transferred to an APIII massspectrometer (Perkin-Elmer, Sciex, Toronto, Canada) and the samplesprayed for approximately 20 minutes. During this time, peptide ionsapparent from the mass spectrum were selected and isolated in turn andfragmented in the collision chamber of the mass spectrometer. From thetandem mass spectra, short stretches of sequence were assembled intopeptide sequence tags (Mann and Wilm, Anal. Chem. 66:4390-99, 1994) andsearched against a protein sequence database or against dbEST usingPeptideSearch.

Three peptides matched the IKK-1 sequence. A1: IIDLGYAK (SEQ ID NO:17);A2: VEVALSNIK (SEQ ID NO:18); A3 SIQLDLER (SEQ ID NO:19). Three otherpeptides matched human EST sequences in dbEST: B1: ALELLPK (SEQ IDNO:20), B2: VIYTQLSK (SEQ ID NO:21), B6: LLLQAIQSFEK (SEQ ID NO:22) allmatch EST clone AA326115. The peptide B4 with the sequence LGTGGFGNVIR(SEQ ID NO:23) was found in clone R06591. After the full-length IKK-2sequence was obtained (as described below) two more peptides B3:ALDDILNLK (SEQ ID NO:24) and B5: DLKPENIVLQQGEQR (SEQ ID NO:25) werefound in the sequence. Peptide A1 is present in both IKK-1 and IKK-2sequences. All the EST clones identified were clearly homologous tohuman and mouse CHUK, a serine/threonine kinase of hitherto unknownfunction. Once the complete coding sequence of IKK-2 was obtained (asdescribed below), all sequenced peptides (apart from two peptidesderived from IKK-1) could be assigned to this protein.

Representative mass spectra are shown in FIGS. 12A and 12B. In FIG. 12A,peaks labeled A were matched to the tryptic peptides of IKK-1 uponfragmentation followed by database searching with peptide sequence tags.Peaks labeled B2, B4, B6 were not found in protein databases but insteadmatched human EST sequences. One more peptide (B1) matching a human ESTclone was observed at m/z 392.2 and is not shown in panel A. In FIG.12B, a continuous series of C-terminal-containing fragments (Y″-ions)was used to construct a peptide sequence tag as shown by boxed letters.Search of this tag resulted in a match to the peptide LLLQALQSFEK (SEQID NO:22) in human EST clone AA326115. Two more peptides, B1 (ALELLPK;SEQ ID NO:20) and B2 (VIYTQLSK; SEQ ID NO:21) were found in the sequenceof the same EST clone.

Full-length human IKK-1 and IKK-2 cDNAs were cloned based on the. humanEST clones, which were obtained from Genome Systems, Inc. (St. Louis,Mo.). The precise nucleotide sequences were determined and used todesign primers to PCR clone human IKK-2 from a human HeLa cell cDNAlibrary (Clontech, Inc., Palo Alto, Calif.). Several IKK-2 cDNA cloneswere isolated and sequenced. Full-length mouse IKK-1 and a partial humanIKK-1 nucleotide sequence was available in the comprehensive database,primers were designed to PCR clone the respective human and mouse IKK-1cDNAs. The partial human IKK-1 coding region was used to probe a HeLacDNA phage library (Stratagene, Inc., La Jolla, Calif.) to obtain thefull-length human IKK-1 cDNA clone using standard procedures.

Sequence analysis of these clones revealed that IKK-1 and IKK-2 wererelated protein serine kinases (51% identity) containing proteininteraction motifs (FIG. 13A). Both IKK-1 and IKK-2 contain the kinasedomain at the N-terminus, and a leucine zipper motif and ahelix-loop-helix motif in their C-terminal regions (FIG. 13A). Northernanalysis indicated that mRNAs encoding IKK-2 were widely distributed inhuman tissues, with transcript sizes of ˜4.5 kb and 6 kb (FIG. 13B). Thedistribution of IKK-1 (CHUK) transcripts has been reported previously(Connely et al., Cell. Mol. Biol. Res. 41:537-49, 1995). IKK-1 and IKK-2mRNAs are constitutively expressed in Jurkat, HeLa and HUVEC cell lines,and their levels are not altered for up to 8 hours following stimulationwith NFκB inducers such as TNFα (HeLa, HTVEC) or anti-CD28 plus PMA(Jurkat).

To further characterize the properties of IKK-1 and IKK-2, recombinantHA-tagged IKK-1 and Flag-tagged IKK-2, either separately or alone, werein vitro transcribed and translated in wheat germ or rabbit reticulocytelysate (Promega, Madison, Wis.). The reactions were performed exactly asdescribed in the manufacturer's protocol. Epitope-tagged IKK-1 and IKK-2then immunoprecipitated with the appropriate anti-tag antibody.Immunoprecipitates containing these proteins phosphorylated IκBα andIκBβ with the correct substrate specificity (i.e., immunoprecipitates ofIKK-1 and IKK-2 phosphorylated both GST-IκBα (FIG. 14A, panel 3) andGST-IκBβ (panel 4), but did not phosphorylate the corresponding S32/36to T mutant protein). IKK-1 expressed in rabbit reticulocyte lysates wasalso capable of autophosphorylation (FIG. 14A, panel 2, lane 1), whereasa kinase-inactive version of IKK-1, in which the conserved lysine 44 hadbeen mutated to methionine, showed no autophosphorylation. In contrastIKK-2, although expressed at equivalent levels in the lysates (panel 1),showed very weak autophosphorylation (panel 2, lane 2).

Expression of the kinase inactive mutants (K to M) of IKK-1 and IKK-2indicate that both play critical roles in NFκB activation asdemonstrated by immunofluorescent studies (FIGS. 14B and 14C). For thesestudies, HeLa cells were transiently transfected with either HA-taggedIKK-1 or Flag-tagged IKK-2. Cells were fixed for 30 minutes withmethanol. For immunofluorescence staining, the cells were incubatedsequentially with primary antibody in PBS containing 10% donkey serumand 0.25% Triton X-100 for 2 hours followed by fluorescein-conjugated orTexas red-conjugated secondary antibody (Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa.; used at 1:500 dilution) for 1 hourat room temperature. The coverslips were rinsed and coverslipped withVectashield (Vector Laboratories, Burlingame, Calif.) before scoring andphotographing representative fields. Primary antibodies used forimmunofluorescence staining included antibodies against Rel A (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.), HA tag (Babco, Berkeley,Calif.) and Flag tag (IBI-Kodak, New Haven, Conn.).

Kinase-inactive versions (K44 to M) of IKK-1 and IKK-2 had no effect onthe subcellular localization of RelA in unstimulated HeLa cells, sinceRelA remained cytoplasmic both in cells expressing the epitope-taggedproteins and in the adjacent untransfected cells (FIGS. 14B and 14C, toppanels). In contrast, both mutant proteins inhibited RelA nucleartranslocation in TNFα-stimulated cells (FIGS. 14B and 14C, bottompanels). The inhibition mediated by the IKK-2 mutant was striking andcomplete (FIG. 14C: compare mutant IKK-2-expressing cells withuntransfected cells in the same field), whereas that mediated by themutant IKK-1 protein, expressed at apparently equivalent levels, wassignificant but incomplete (FIG. 14B). This difference in the functionalactivities of the two mutant kinases may point to distinct roles forthese two kinases in NFκB activation.

The presence of the leucine zipper and helix-loop-helix motif in IKK-1and IKK-2 suggested that they interacted functionally with otherproteins in the signalsome. An obvious possibility was that the proteinsformed hetero- or homodimers with one another. HA-tagged IKK-1 andFLAG-tagged IKK-2 were translated in rabbit reticulocyte lysates, eitheralone or together, and then immunoprecipitated with antibodies to theappropriate epitope tags. This experiment demonstrated clearly thatIKK-2 was present in IKK-1 immunoprecipitates (FIG. 15A, lane 3) andvice versa (lane 4), suggesting that these proteins either associateddirectly or via adapter proteins/IKK signalsome components present inthe rabbit reticulocyte lysates. In contrast, however, there was noevidence for association of IKK-1 and IKK-2 that had been cotranslatedin wheat germ lysates (FIG. 15B), suggesting that the proteins did notheterodimerize directly. When full-length IKK-1 was translated togetherin wheat germ extracts with a truncated version of IKK-1 that stillpossessed the protein interaction motifs, there was also no evidence ofassociation, suggesting that IKK-1 was also not capable of forminghomodimers under these conditions.

Both IKK-1 and IKK-2 kinases were active when expressed in wheat germextracts, since they were capable of autophosphorylation, but they wereinactive with respect to phosphorylation of IκB substrates. Since bothautophosphorylation and substrate phosphorylation were intact in rabbitreticulocyte lysates, there appeared to be a direct correlation betweenthe association of IKK-1 and IKK-2 into a higher order protein complexand the presence of specific IκB kinase activity in IKK-1 and IKK-2immunoprecipitates. This higher order complex is most likely the IKKsignalsome itself. Indeed, immunoprecipitation of rabbit reticulocytelysates with anti-MKP-1 antibody pulls down a low level of active IκBkinase activity characteristic of the IKK signalsome.

It is clear that the IKK signalsome contains multiple protein componentsin addition to IKK-1 and IKK-2 (FIG. 11B). Some of these may be upstreamkinases such as MEKK-1 (Chen et al., Cell 84:853-62, 1996) or NIK(Malinin, et al., Nature 385:540-44, 1997); others may be adapterproteins that mediate the IKK-1:IKK-2 interaction. Indeed MEKK-1copurifies with IKK signalsome activity (FIG. 1C), and two othersignalsome proteins have been functionally identified. The proteincrossreactive with anti-MKP-1 is an intrinsic component of the IKKsignalsome kinases, since the IκB kinase activity coprecipitated withthis antibody is stable to washes with 2-4 M urea. Moreover, both IKK-1immunoprecipitates and MKP-1 immunoprecipitates containing the IKKsignalsome (FIG. 8C) contain an inducible RelA kinase whose kinetics ofactivation parallel those of the IκB kinase in the sameimmunoprecipitates. Another strong candidate for a protein in thesignalsome complex is the E3 ubiquitin ligase that transfersmultiubiquitin chains to phosphorylated IκB (Hershko et al., Annu. Rev.Biochem. 61:761-807, 1992).

These results indicate that IKK-1 and IKK-2 are functional kinaseswithin the IKK signalsome, which mediate IκB phosphorylation and NFκBactivation. Appropriate regulation of IKK-1 and IKK-2 may require theirassembly into a higher order protein complex, which may be a heterodimerfacilitated by adapter proteins, the complete IKK signalsome, or someintermediate subcomplex that contains both IKK-1 and IKK-2.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

1. An isolated polynucleotide encoding a protein comprising thepolypeptide of SEQ ID NO:9.
 2. The isolated polynucleotide of claim 1 orcomplement thereof, wherein said polynucleotide or complement is a DNAmolecule.
 3. The isolated polynucleotide according to claim 1 comprisingSEQ ID NO:8, or the complement of said polynucleotide.
 4. The isolatedpolynucleotide according to claim 1 consisting essentially of SEQ IDNO:8, or the complement of said polynucleotide.
 5. A recombinantexpression vector comprising the polynucleotide according to claim
 1. 6.An isolated host cell transformed or transfected with the expressionvector of claim
 5. 7. The isolated host cell according to claim 6,wherein the host cell is selected from the group consisting of bacteria,yeast, baculovirus infected insect cells and mammalian cells.
 8. Anisolated polynucleotide comprising a polynucleotide sequence encoding apolypeptide comprising SEQ ID NO:9, or the complement of said isolatedpolynucleotide.
 9. The isolated polynucleotide of claim 8 that is a DNAmolecule.
 10. The isolated polynucleotide of claim 8 comprising thecoding region of SEQ ID NO:8.
 11. The isolated polynucleotide of claim1, wherein said polypeptide is a fusion protein.
 12. The isolatedpolynucleotide of claim 1, wherein said polypeptide comprises a peptidetag useful for purification of said polypeptide.
 13. The isolatedpolynucleotide of claim 8, wherein said polypeptide is a fusion protein.14. The isolated polynucleotide of claim 8, wherein said polypeptidecomprises a peptide useful for purification of the polypeptide.
 15. Anisolated polypeptide that is a catalytically inactive variant of thepolypeptide of SEQ ID NO:9, wherein the variant has the sequence of SEQID NO:9 with a single substitution of the lysine residue at position 44by a methionine residue.
 16. An isolated polynucleotide encoding acatalytically inactive variant of the polypeptide of SEQ ID NO:9,wherein the variant has the sequence of SEQ ID NO:9 with a singlesubstitution of the lysine residue at position 44 by a methionineresidue.
 17. An isolated DNA molecule comprising SEQ ID NO:
 7. 18. Anisolated DNA molecule consisting of SEQ ID NO: 7.